The ability to efficiently, rapidly and unambiguously analyze polymorphisms in the nucleic acid sequences of a gene of interest plays an important role in the development of molecular diagnostic assays, the applications of which includes genetic testing, carrier screening, genotyping or genetic profiling, and identity testing. For example, it is the objective of genetic testing and carrier screening to determine whether mutations associated with a particular disease are present in a gene of interest. The analysis of polymorphic loci, whether or not these comprise mutations known to cause disease, generally provides clinical benefit, as for example in the context of pharmacogenomic genotyping or in the context of HLA molecular typing, in which the degree of allele matching in the HLA loci of transplant donor and prospective recipient is determined in context of allogeneic tissue and bone marrow transplantation.
The multiplexed analysis of polymorphisms while desirable in facilitating the analysis of a high volume of patient samples, faces a considerable level of complexity which will likely increase as new polymorphisms, genetic markers and mutations are identified and must be included in the analysis. The limitations of current methods to handle this complexity in a multiplexed format of analysis so as to ensure reliable assay performance while accommodating high sample volume, and the consequent need for novel methods of multiplexed analysis of polymorphisms and mutations is the subject of the present invention. By way of example, the genetic loci encoding the Cystic Fibrosis Transmembrane Conductance (CFTR) channel and Human Leukocyte Antigens (HLA) are analyzed by the methods of the invention. Cystic fibrosis (CF) is one of the most common recessive disorders in Caucasians with a rate of occurrence in the US of 1 in 2000 live births. About 4% of the population carry one of the CF mutations. The CFTR gene is highly variable: more than 900 mutations have been identified to date (see the website at found at server name genet.sickkids.on with domain name ca, at resource ID cftr, which is incorporated herein by reference). The characterization of the CFTR gene provides the key to the molecular diagnosis of CF by facilitating the development of sequence-specific probes (Roments et al., 1989; Riordan, et al., 1989; Kerem et al., 1989, each of which is incorporated herein by reference). The National Institutes of Health (NIH)—sponsored consensus development conference recommended carrier screening for CFTR mutations for adults with a positive family history of CF (NIH 1997). The committee on carrier screening of the American College of Medical Genetics (ACMG) has recommended for use in general population carrier screening a pan-ethnic mutation panel that includes a set of 25 disease-causing CF mutations with an allele frequency of >0.1% in the general population of United States (see the Federation of American Societies of Experimental Biology website (domain name .org) at resource ID genetics/acmg, which is incorporated herein by reference). The mutations in the ACMG panel also include the most common mutations in Ashkenazi Jewish and African-American populations.
Several methods have been described for the detection of CFTR mutations including the following: denaturing gradient gel electrophoresis (Devoto et al., 1991); single strand conformation polymorphism analysis (Plieth et al., 1992); RFLP (Friedman et al., 1991); amplification with allele-specific primers (ASPs) (Gremonesi et al., 1992), and probing with allele specific oligonucleotides (ASO) (Saiki et al., 1986). A widely used method involves PCR amplification followed by blotting of amplified target strands onto a membrane and probing of strands with oligonucleotides designed to match either the normal (“wild type”) or mutant configuration. Specifically, multiplex PCR has been used in conjunction with ASO hybridization in this dot blot format to screen 12 CF mutations (Shuber et al., 1993). In several instances, arrays of substrate-immobilized oligonucleotide probes were used to facilitate the detection of known genomic DNA sequence variations (Saiki, R K et al., 1989) in a “reverse dot blot” format An array of short oligonucleotides synthesized in-situ by photolithographic processes was used to detect known mutations in the coding region of the CFTR gene (Cronin, M T., et al., 1996). Primer extension using reverse transcriptase has been reported as a method for detecting the A508 mutation in CFTR (Pastinen, T., 2000). This approach was described as early as 1989 (Wu, D. Y. et al, Proc. Natl. Acad. Sci. USA. 86:2757-2760 (1989), Newton, C. R. et al, Nucleic Acids Res. 17:2503-2506 (1989)). As further discussed herein below, while providing reasonable detection in a research laboratory setting, these methods require significant labor, provide only slow turnaround, offer only low sample throughput, and hence require a high cost per sample.
In connection with the spotted microarrays, several methods of spotting have been described, along with many substrate materials and methods of probe immobilization. However, the spotted arrays of current methods exhibit not only significant array-to-array variability but also significant spot-to-spot variability, an aspect that leads to limitations in assay reliability and sensitivity. In addition, spotted arrays are difficult to miniaturize beyond their current spot dimensions of typically 100 μm diameter on 500 μm centers, thereby increasing total sample volumes and contributing to slow assay kinetics limiting the performance of hybridization assays whose completion on spotted arrays may require as much as 18 hours. Further, use of spotted arrays involve readout via highly specialized confocal laser scanning apparatus. In an alternative approach, oligonucleotide arrays synthesized in-situ by a photolithographic process have been described. The complexity of array fabrication, however, limits routine customization and combines considerable expense with lack of flexibility for diagnostic applications.
The major histocompatibility complex (MHC) includes the human leukocyte antigen (HLA) gene complex, located on the short arm of human chromosome six. This region encodes cell-surface proteins which regulate the cell-cell interactions underlying immune response. The various HLA Class I loci encode 44,000 dalton polypeptides which associate with 13-2 microglobulin at the cell surface and mediate the recognition of target cells by cytotoxic T lymphocytes. HLA Class II loci encode cell surface heterodimers, composed of a 29,000 dalton and a 34,000 dalton polypeptide which mediate the recognition of target cells by helper T lymphocytes. HLA antigens, by presenting foreign pathogenic peptides to T-cells in the context of a “self” protein, mediate the initiation of an immune response. Consequently, a large repertoire of peptides is desirable because it increases the immune response potential of the host. On the other hand, the correspondingly high degree of immunogenetic polymorphism represents significant difficulties in allotransplantation, with a mismatch in HLA loci representing one of the main causes of allograft rejection. The degree of allele matching in the HLA loci of a donor and prospective recipient is a major factor in the success of allogeneic tissue and bone marrow transplantation.
The HLA-A, HLA-B, and HLA-C loci of the HLA Class I region as well as the HLA-DRB, HLA-DQB, HLA-DQA, HLA-DPB and HLA-DPA loci of the HLA Class II region exhibit an extremely high degree of polymorphism. To date, the WHO nomenclature committee for factors of the HLA system has designated 225 alleles of HLA A (HLA A*0101, A*0201, etc.), 444 alleles of HLA-B, and 111 alleles of HLA-C, 358 HLA-DRB alleles, 22 HLA-DQA alleles, 47 HLA-DQB alleles, 20 HLA-DPA alleles and 96 HLA-DPB alleles (See IMGT/HLA Sequence Database, found at server name ebi.ac with domain name uk80, at resource ID imgt/h1a/index.html and Schreuder, G. M. Th. et al, Tissue Antigens. 54:409-437 (1999)), both of which are hereby incorporated by reference.
HLA typing is a routine procedure that is used to determine the immunogenetic profile of transplant donors. The objective of HLA typing is the determination of the patient's allele configuration at the requisite level of resolution, based on the analysis of a set of designated polymorphisms within the genetic locus of interest. Increasingly, molecular typing of HLA is the method of choice over traditional serological typing, because it eliminates the requirement for viable cells, offers higher allelic resolution, and extends HLA typing to Class II for which serology has not been adequate (Erlich, H. A. et al, Immunity 14:347-356 (2001)).
One method currently applied to clinical HLA typing uses the polymerase chain reaction (PCR) in conjunction with sequence-specific oligonucleotide probes (SSO or SSOP), which are allowed to hybridize to amplified target sequences to produce a pattern as a basis for HLA typing.
The availability of sequence information for all available HLA alleles has permitted the design of sequence-specific oligonucleotides (SSO) and allele-specific oligonucleotides (ASO) for the characterization of known HLA polymorphisms as well as for sequencing by hybridization (Saiki, R. K. Nature 324:163-166 (1986), Cao, K. et al, Rev Immunogenetics, 1999: 1: 177-208).
In one embodiment of SSO analysis, also referred to as a “dot blot format”, DNA samples are extracted from patients, amplified and blotted onto a set of nylon membranes in an 8×12 grid format. One radio-labeled oligonucleotide probe is added to each spot on each such membrane; following hybridization, spots are inspected by autoradiography and scored either positive (1) or negative (0). For each patient sample, the string of 1's and 0's constructed from the analysis of all membranes defines the allele configuration. A multiplexed format of SSO analysis in the “reverse dot blot format” employs sets of oligonucleotide probes immobilized on planar supports (Saiki, R. et al, Immunological Rev. 167: 193-199 (1989), Erlich, H. A. Eur. J. Immunogenet. 18: 33-55 (1991)).
Another method of HLA typing uses the polymerase-catalyzed elongation of sequence-specific primers (SSPs) to discriminate between alleles. The high specificity of DNA polymerase generally endows this method with superior specificity. In the SSP method, PCR amplification is performed with a specific primer pair for each polymorphic sequence motif or pair of motifs and a DNA polymerase lacking 3′→5′ exonuclease activity so that elongation (and hence amplification) occurs only for that primer whose 3′ terminus is perfectly complementary (“matched”) to the template. The presence of the corresponding PCR product is ascertained by gel electrophoretic analysis. An example of a highly polymorphic locus is the 280 nt DNA fragment of the HLA class II DR gene which features a high incidence of polymorphisms
HLA typing based on the use of sequence-specific probes (SSP), also referred to as phototyping (Dupont, B. Tissue Antigen. 46: 353-354 (1995)), has been developed as a commercial technology that is in routine use for class I and class II typing (Bunce, M. et al, Tissue Antigens. 46:355-367 (1995), Krausa, P and Browning, M. J., Tissue Antigens. 47: 237-244 (1996), Bunce, M. et al, Tissue Antigens. 45:81-90 (1995)). However, the requirement of the SSP methods of the prior art for extensive gel electrophoretic analysis for individual detection of amplicons represents a significant impediment to the implementation of multiplexed assay formats that can achieve high throughput. This disadvantage is overcome by the methods of the present invention.
In the context of elongation reactions, highly polymorphic loci and the effect of non-designated polymorphic sites as interfering polymorphisms were not considered in previous applications, especially in multiplexed format. Thus, there is a need to provide for methods, compositions and processes for the multiplexed analysis of polymorphic loci that would enable the detection of designated while accommodating the presence of no-designated sites and without interference from such non-designated sites.